1264
Biochemistry 2004, 43, 1264-1275
Removal of PsaF Alters Forward Electron Transfer in Photosystem I: Evidence for
Fast Reoxidation of QK-A in Subunit Deletion Mutants of Synechococcus sp. PCC
7002†
Art van der Est,*,‡ Alfia I. Valieva,‡,§ Yuri E. Kandrashkin,‡,| Gaozhong Shen,⊥ Donald A. Bryant,⊥ and
John H. Golbeck⊥
Department of Chemistry, Brock UniVersity, 500 Glenridge AVenue, St. Catharines, Ontario, Canada L2S 3A1, Kazan Institute
of Biochemistry and Biophysics and Kazan Physical Technical Institute, RAS, Kazan, Russia, and Department of Biochemistry
and Molecular Biology, The PennsylVania State UniVersity, UniVersity Park, PennsylVania 16802
ReceiVed August 11, 2003; ReVised Manuscript ReceiVed December 5, 2003
ABSTRACT: Recent studies of point mutations in photosystem I have suggested that the two kinetic phases
of phylloquinone reoxidation represent electron transfer in the two branches of cofactors. This interpretation
implies that changes in the relative amplitudes of the two kinetic phases represent a change in the extent
of electron transfer in the two branches. Using time-resolved electron paramagnetic resonance (EPR),
this issue is investigated in subunit deletion mutants of Synechococcus sp. PCC 7002. The spin-polarized
EPR signals of P700+A1- and P700+FeS-, both at room temperature and in frozen solution, are altered by
deletion of PsaF and/or PsaE, and the differences from the wild type are much more pronounced in PS
I complexes isolated from the mutants using Triton X-100 rather than n-dodecyl β-D-maltopyranoside.
The changes in the transient EPR data for the mutant complexes are consistent with a significant fraction
of reaction centers showing (i) faster electron transfer from A1- to FX, (ii) slower forward electron transfer
from A0- to A1, and (iii) slightly altered quinone hyperfine couplings, possibly as a result of a change in
the hydrogen bonding. The fraction of fast electron transfer and its dependence on the isolation procedure
are estimated approximately from simulations of the room temperature EPR data. The results are discussed
in terms of possible models for the electron transfer. It is suggested that the detergent-induced fraction of
fast electron transfer is most likely due to alteration of the environment of the quinone in the PsaA branch
of cofactors and is not the result of a change in the directionality of electron transfer.
According to the identity of their terminal acceptors,
photosynthetic reaction centers are divided into two classes
referred to as type I and type II. The general arrangements
of the cofactors in the electron-transfer chain are similar in
type I (photosystem I (PS I)1 and reaction centers of green
sulfur bacteria and heliobacteria) and in type II (photosystem
II (PS II) and purple bacteria) reaction centers. Both types
have two nearly symmetric branches of acceptors extending
across the membrane from the primary donor to a mobile
electron carrier on the acceptor side. However, the nature
† This work was supported by grants to A.v.d.E. from the NSERC,
CFI, and OIT and to J.H.G. (MCB-0117079) and to D.A.B. (MCB0077586) from the U.S. National Science Foundation.
* To whom correspondence should be addressed. Phone: 1 (905)
688-5550. Fax: 1 (905) 682-9020. E-mail: [email protected].
‡ Brock University.
§ Kazan Institute of Biochemistry and Biophysics, RAS.
| Kazan Physical Technical Institute, RAS.
⊥
The Pennsylvania State University.
1
Abbreviations: PS I, photosystem I; PS II, photosystem II, P700,
primary donor in PS I, A0, primary chlorophyll acceptor in PS I; A1,
quinone acceptor in PS I; FX, FA, and FB, iron-sulfur clusters in PS I;
FeS, unpecified iron-sulfur cluster; PsaA, PsaB, etc., protein subunits
of the PS I complex; QK-A, QK-B, phylloquinones bound to PsaA and
PsaB; β-DM, n-dodecyl-β-D-maltopyranoside; WT, wild type; ∆psaE,
∆psaF, ∆psaE/∆psaF, subunit deletion mutants; EPR, electron paramagnetic resonance; ENDOR, electron nuclear double resonance; E,
emission; A, absorption.
of the mobile acceptor is considerably different in the two
types of reaction centers. In type II, the quinone associated
with the B-branch of cofactors acts as the terminal acceptor
and as a mobile electron carrier. Because of this, the initial
charge separation is strongly biased toward the A-branch of
cofactors. In contrast, in type I reaction centers such as PS
I, the two branches converge at the acceptor FX, and as a
result, the mobile electron carrier, ferredoxin or flavodoxin,
is not associated with either of the two branches. This raises
the question of whether both of the quinones are active in
electron transfer in PS I or one of the quinones plays a
different, unrecognized role.
Recently, this question has been addressed by studying
the kinetics of quinone reoxidation using various forms of
time-resolved spectroscopy (1-5). In particular, optical
studies of the absorbance changes in the near-UV have shown
that in many species the electron-transfer kinetics from A1
to FX has two components that can be affected by a number
of factors (see refs 6 and 7 for reviews). In spinach PS I
complexes, the reoxidation of A1 has been found to occur
with t1/2 values of about 25 and 150 ns (8), while in
cyanobacterial PS I, initial measurements suggested that the
kinetics were monophasic with a t1/2 of about 200 ns (911). However, later experiments with higher time resolution
revealed a minor fraction with a t1/2 of 6.6 ns (12). The slow
10.1021/bi035431j CCC: $27.50 © 2004 American Chemical Society
Published on Web 01/16/2004
Forward Electron Transfer in Photosystem I
kinetic phase is strongly temperature dependent (13), while
the fast phase is virtually independent of temperature (14).
Below the glass transition temperature of the protein, forward
transfer past A1 is blocked in approximately half of the
reaction centers and stable formation of P+(FA/FB)- occurs
in the other half. At present it is unknown whether a
correlation exists between the two fractions observed at low
temperature and the two kinetic phases observed at higher
temperature. An interesting feature of the two phases is that
in spinach the relative amplitudes are very dependent on the
method used to isolate PS I (8); a larger fraction of fast
electron transfer is observed in complexes isolated using
strong detergents. When Triton X-100 and β-mercaptoethanol
are used, the fast phase dominates, while milder treatments
give roughly equal amounts of the two phases, and when a
Yeda press is used (i.e., no detergent), the fast kinetic phase
is found to account for only 30% of the electron transfer.
These results suggest that the fast phase might be a
preparation-induced artifact. However, Joliot and Joliot (15)
recently used optical spectroscopy with nanosecond time
resolution to study the rate of forward electron transfer from
A1 to FX in whole cells of a mutant strain of Chlorella
sorokiniana lacking PS II and the peripheral antennae. They
observed two phases with approximately equal amplitudes
and t1/2 values of 18 and 160 ns, clearly showing that the
biphasic kinetics is an inherent property of PS I electron
transfer and not a preparation-induced artifact.
Two hypotheses have been proposed to explain the origin
of the two kinetic phases of quinone reoxidation (2, 15). The
first hypothesis suggests that the two kinetic phases represent
electron transfer in the two branches of cofactors, while the
second hypothesis assigns the two phases to two populations
of PS I that differ structurally. The bidirectional model is
supported primarily by point mutation studies of the eukaryote Chlamydomonas reinhardtii (1-3). These studies involved mutation of the tryptophan residue present in both
branches, which is π-stacked with the respective phylloquinone. Mutation of the tryptophan on the PsaB side affects
only the fast component, slowing the half-life of electron
transfer from ∼20 to 73 ns, whereas the corresponding
mutation on the PsaA side affects only the slower component,
slowing it from ∼200 to ∼490 ns. Combined mutations in
both sides alter both the fast and slow kinetic components.
A more extensive study of mutants of the cyanobacterium
Synechocystis sp. PCC 6803 (4, 5) shows similar trends. The
slow kinetic phase could clearly be associated with the PsaA
branch of cofactors, while the correlation between mutations
in the PsaB branch and changes in the fast kinetic phase,
although present, was not as strong. Electron transfer along
the PsaA branch is supported by low temperature and timeresolved EPR data (1, 5) and is consistent with earlier
photoaccumulation studies on subunit deletion mutant strains
of Synechococcus sp. PCC 7002 (16). In the latter, it was
shown that when the psaE and psaF genes were deleted and
Triton X-100 was used to solubilize PS I, A0- was photoaccumulated at the expense of A1-. PsaE and PsaF were the
only subunits for which such an effect was observed, and
these subunits are located on the stromal side of the
membrane on one side of the PS I complex. It was therefore
suggested that the EPR-visible A1- signal is produced by
Biochemistry, Vol. 43, No. 5, 2004 1265
the quinone2 in the branch associated with the subunit closest
to PsaE and PsaF (17). The difference in photoaccumulation
behavior was explained by postulating that, in the absence
of PsaE and PsaF, the detergent is able to open a water
channel to the quinone, allowing it to be protonated and
hence doubly reduced (16). This, in turn, allows A0- to be
photoaccumulated. Although the quinone associated with the
branch closer to PsaF was implicated, it was not known at
the time which of the two subunits, PsaA or PsaB, corresponded to this branch. With the determination of the X-ray
structure at 2.5 Å resolution, it is now known that the PsaA
branch is closer to PsaF. This asymmetry in the location of
the subunits is displayed in Figure 1, which shows two
different views of the PS I complex that highlight the relative
positions of the cofactor branches and the PsaE and PsaF
subunits. The views, approximately along the membrane
normal (crystallographic c-axis) from the stromal side of the
membrane and parallel to the membrane plane, illustrate most
clearly that these two subunits are much closer to the PsaA
branch than the PsaB branch.
These results raise a number of interesting issues. In the
unidirectional model of electron transfer, a change in the
ratio of the two phases implies a change in the relative
amounts of the two conformations associated with the two
phases of electron transfer. In the bidirectional model, it
implies a change in the fraction of electrons that are
transferred along the two branches. Thus, the results showing
that the use of strong detergents increases the amount of fast
electron transfer in spinach preparations (8) imply that the
detergent either changes the conformation of the protein near
the active quinone or increases the fraction of electrons that
are transferred along the PsaB branch. In the latter case, the
detergent would need to affect P700 and/or A0 because the
ratio of electron transfer along the two branches is determined
during the initial charge separation. Thus, a better understanding of the detergent effects on electron transfer in PS I
should provide additional information for distinguishing
between the two possible models.
In spinach PS I preparations, strong detergent treatments
lead to reduced amounts of proteins of masses of 8, 16.5,
and 20-22 kDa (18), a molecular mass range which includes
the PsaE, PsaF, and PsaD proteins. Since these types of
preparations typically show larger amounts of fast electron
transfer to FX, it is possible that the change in the ratio of
the two phases is related to the loss of the peripheral subunits.
Here, we investigate this possibility by studying the electrontransfer kinetics in peripheral subunit deletion mutants of
cyanobacteria that show altered photoaccumulation behavior
when PS I is isolated using Triton X-100. The motivation
for these experiments is to test the proposal (16) that the
combined effects of the detergent and mutation alter the
protein environment of the quinone associated with the PsaA
branch by correlating the changes in the photoaccumulation
behavior with changes in the electron-transfer kinetics.
We use transient EPR spectroscopy for these experiments
because it allows both the electron-transfer kinetics and the
interaction of the quinone with its environment to be studied
simultaneously (see refs 19-25 for a series of reviews). A
2
Synechococcus sp. PCC 7002 has been shown to contain menaquinone-4 rather than phylloquinone (Y. Sakuragi and D. A. Bryant,
unpublished results).
1266 Biochemistry, Vol. 43, No. 5, 2004
FIGURE 1: Location of the PsaE and PsaF subunits in PS I from
the 2.5 Å resolution X-ray structure (69) (PDB entry 1JB0). The
complex is shown as viewed from two different perspectives. Top:
view from the stromal side of the membrane along a direction
roughly parallel to the membrane normal. Bottom: view from a
direction roughly parallel to the membrane plane. The backbones
of the PsaE and PsaF proteins are shown along with the electrontransfer cofactors. The figure was constructed using the program
MOLMOL (83).
potential drawback of this method is that the spectrometer
response time is typically ∼50 ns and hence too slow to
directly resolve fast electron-transfer steps. However, the
presence of a short-lived precursor can be revealed indirectly
through its influence on the spin polarization of subsequent
radical pairs (26-30). This technique has been well established by studying the electron transfer from pheophytin to
the quinone in modified reaction centers of purple bacteria
(27, 28, 31-36). However, until fairly recently, similar
studies in PS I were hampered by the lack of reliable
magnetic interaction parameters, distances, and geometries
for the cofactors. Over the past several years these parameters
have become available from studies using high-field EPR
(37-41), echo envelope modulation (42-48), pulsed and
CW-ENDOR (49-54), quantum beats (55, 56), and PS I
van der Est et al.
single crystals (43, 57-61). Recently, we investigated the
room temperature polarization patterns of the sequential
radical pair states P700+A1- and P700+FeS- in cyanobacteria
and showed that they are reproduced very well in simulations
in which all of the necessary parameters are fixed using
independent experimental data (30, 62). These simulations
are simplified by the fact that the small amplitude and short
lifetime of the fast phase of electron transfer from A1- to
FX in cyanobacteria make its contribution to the polarization
patterns very small. However, this fraction can also be taken
into account as shown by corresponding simulations of PS
I from C. rheinhardtii in which a significant influence of
the fast phase is observed (63). In native PS I preparations,
the primary radical pair is too short-lived to have a significant
effect on the spin polarization. However, in green sulfur
bacteria and heliobacteria, the lifetime of P+A0- is more than
an order of magnitude longer and effects similar to those
observed in modified purple bacterial reaction centers (27,
28, 31-36) are seen (64). Again, the polarization patterns
can be reproduced by taking the spin dynamics into account
and by assuming a reasonable set of magnetic parameters
(62, 65).
All of these studies show that transient EPR is sensitive
to the presence of short-lived precursors, and in wellcharacterized systems such as PS I and purple bacterial
reaction centers, it can be used to estimate properties of such
precursors even though they are not resolved kinetically.
Here, we use this approach to investigate the influence of
the deletion mutations and detergent isolation on the electrontransfer kinetics in cyanobacterial PS I. It is important to
stress that this method can be expected to give accurate
qualitative information about changes in the electron-transfer
kinetics but not numerical values of the lifetimes of the shortlived radical pairs. We will show that the combined effect
of deletion of PsaE and PsaF and isolation of PS I using
Triton X-100 is to induce a much larger fraction of fast
electron transfer to the iron sulfur clusters. Because PsaE
and PsaF are located close to PsaA, we propose that this is
due to changes in the electron transfer in the PsaA branch
of cofactors and not due to electrons being redirected along
the PsaB branch.
MATERIAL AND METHODS
Mutant strains of Synechococcus sp. PCC 7002 devoid of
selected peripheral photosystem I subunits were prepared and
grown as described in ref 16. Three mutants were studied in
which (i) the psaE gene was interrupted (∆psaE), (ii) the
psaF gene was interrupted (∆psaF), and (iii) the psaE gene
and the psaF gene were both interrupted (∆psaE/∆psaF).
PS I trimers from wild-type and mutant strains were isolated
using either 30 mM n-dodecyl-β-D-maltopyranoside (β-DM)
or 1% (v/v) Triton X-100 to solubilize the thylakoid
membranes as described previously (16). Trimeric PS I
complexes were prepared at a chlorophyll concentration of
1.5-2 mg mL-1 in 50 mM Tris, pH 6.8, containing 15%
(v/v) glycerol and 0.03% (w/v) β-DM or 1% (v/v) Triton
X-100. To reduce P700, 5 µM phenazine methosulfate (PMS)
and 20 mM sodium ascorbate were added to the PS I
complexes prior to the measurements.
Transient EPR time/field data sets were recorded at both
room temperature and 135 K using a modified Bruker ER
Forward Electron Transfer in Photosystem I
FIGURE 2: Room temperature transient EPR spectra of PS I
complexes from the wild type and mutants lacking PsaE and/or
PsaF. Left: PS I complexes isolated using Triton X-100. Right:
PSI complexes isolated using β-DM. From top to bottom (i) wild
type, (ii) ∆psaE mutant, (iii) ∆psaF mutant, (iv) ∆psaE/∆psaF
double mutant. The data are plotted such that absorptive signals
(A) are positive and emissive signals (E) are negative. The two
arrows labeled a and b indicate the positions of the transients shown
in Figure 3. The spectra have been extracted from the data sets by
integrating the signal intensity in time windows centered at 100 ns
(solid line) and 1.2 µs (dashed line) after the laser flash. For the
wild-type PS I complex, the solid curves are dominated by P700+A1while the dashed curves are due to P700+FX-.
200D-SRC X-band spectrometer which is described in detail
elsewhere (5, 66).
RESULTS
Room Temperature Transient EPR Results. Figure 2 shows
room temperature, spin-polarized transient EPR spectra of
PS I from the mutants and wild type. The spectra are taken
at 100 ns (solid curves) and 1.2 µs (dashed curves) after the
laser flash, and the two arrows labeled a and b indicate the
field positions corresponding to the transients shown in
Figure 3. Figures 2 and 3 also show a comparison of
transients from PS I complexes isolated using Triton X-100
and β-DM displayed on the left and right, respectively. In
all of the figures, positive signals represent absorptive spin
polarization (A) and negative signals represent emissive
polarization (E). As can be seen in Figure 2 and in the top
part of Figure 3, an emissive signal is observed at position
a, while at position b (Figure 3, bottom) an absorptive
contribution at early time changes to an emissive signal at
later time. The emissive signal at position a and the early
absorptive contribution at position b are due to the radical
pair P700+A1-, which dominates the spectra of the wild type
(WT) in Figure 2 at 100 ns (solid curves). The emissive part
of the transient at position b, on the other hand, is due to
P700+ in the state P700+FeS-, which gives rise to the spectra
at 1.2 µs in Figure 2. Although it is known (11, 67) that
electron transfer from A1 proceeds via FX, we use FeS to
represent one of the three iron-sulfur clusters and allow for
the possibility that electron transfer from FX to FA/FB is faster
Biochemistry, Vol. 43, No. 5, 2004 1267
than from A1 to FX. Thus, the decay of the signals at position
a and the transition from absorption to emission at position
b are dominated by the rate of electron transfer from A1- to
FX. As is apparent, the transients at position a are all governed
by the same lifetime, and fits of the data sets, which will be
discussed below, yield an average value of 290 ns for the
electron-transfer lifetime. The transients in the bottom part
of Figure 3 are plotted with the same normalization as in
the top part of the figure. Clearly, the relative amplitudes of
the absorptive and emissive polarization differ significantly
among the four samples isolated using Triton X-100 (Figure
2, left), while the four β-DM samples show much smaller
changes. The differing intensities of the emissive polarization
in the Triton X-100 samples are consistent with differing
contributions from P700+FeS- at early time. Since the
emissive contribution is strongest in the complexes isolated
from the double mutant using Triton X-100, the results
suggest that the combined effect of the mutations and Triton
X-100 is to induce fast electron transfer to FX in a significant
fraction of the reaction centers. This is seen most clearly in
the spectra in Figure 2, which show differing amounts of
emissive polarization at early time in the Triton X-100
samples (Figure 2, left, solid curves). The most dramatic
effect is found for the ∆psaE/∆psaF double mutant, which
has a completely emissive spectrum at 100 ns (Figure 2,
bottom left, solid curve). The spectra at 1.2 µs also differ
slightly among the Triton X-100 samples. In the wild type,
this spectrum is almost purely emissive, while for the mutant
samples an absorptive feature on the upfield end of the
spectra is seen, which is again strongest in the ∆psaE/∆psaF
double mutant. There are several factors that could lead to
such changes. The polarization of P700+ in the state P700+FeSdepends on the spin polarization pattern, lifetimes, relative
orientations, and magnetic parameters of P700+, A0-, and A1-.
For example, as discussed in ref 65, an absorptive feature
on the upfield end of the P700+FX- spectrum can occur if
singlet-triplet mixing takes place in the state P700+A0-. Such
effects are also well-known in purple bacteria when the
lifetime of P865+I- is increased (34-36). Possible changes
in the features of the spectrum of P700+A1- are difficult to
study at room temperature because this requires separating
the contributions from P700+A1- and P700+FeS- in the data
sets. Although this is possible using kinetic modeling (11),
it is more convenient to measure the spectrum of P700+A1at low temperature, where no contribution from P700+FeSis observed.
Low-Temperature Transient EPR Results. Figure 4 shows
transient EPR spectra of the same samples taken at 135 K.
Under these conditions the spectra are due to P700+A1- in
those reaction centers in which electron transfer past A1 is
blocked. Recent optical studies show that the fast component
of electron transfer to FX is independent of temperature in a
minor fraction of reaction centers (14), suggesting that an
electron-transfer cycle involving P700+FX- may also occur
to some extent under the conditions used for Figure 4.
However, it has also been shown that the low-temperature
transient EPR spectra of PS I from the wild type and from
mutants lacking FX, FA, and FB are identical (68). Hence,
any contribution from P700+FeS- is negligible at low temperature. The comparison shown in Figure 4 reveals that the
shape of the P700+A1- spectra is also altered in the deletion
mutant complexes isolated using Triton X-100 (Figure 4,
1268 Biochemistry, Vol. 43, No. 5, 2004
van der Est et al.
FIGURE 3: Comparison of EPR transients taken at the two different field positions indicated by the two arrow labels a and b in Figure 2.
The transients taken at position a (top) are dominated by the A1- contribution to P700+A1-, while the transients at position b show an
absorptive (positive) contribution from P700+A1- at early times and an emissive signal (negative) due to P700+FeS- at later times. The traces
on the left side of the figure are from PS I complexes isolated with Triton X-100, and those on the right are from complexes isolated with
β-DM.
FIGURE 4: Spin-polarized transient EPR spectra of P700+A1- at 135
K. The spectra are the integrated signal intensity taken in a time
window from 0.4 to 0.8 µs after the laser flash. As in Figures 2
and 3, the spectra on the left of the figure are from PS I complexes
isolated with Triton X-100 and those on the right are from
complexes isolated with β-DM.
left). Similar changes are also observed in the β-DM samples
(Figure 4, right), but they are much less pronounced. The
main difference between the spectra of the wild-type and
mutant PS I complexes is a change in the relative intensities
of the emissive and absorptive features (see the discussion
of Figure 5, below). In addition there is a small but
reproducible loss of the shoulders (indicated by arrows in
the wild-type spectrum) attributed to hyperfine coupling to
the methyl group of the quinone. Recently, it has been shown
that point mutations in the phylloquinone binding site in the
PsaA branch affect the low-temperature spin polarization
patterns in both green algae (1) and cyanobacteria (5) while
the corresponding mutations in the PsaB branch do not,
demonstrating that the quinone observed in low-temperature
transient EPR experiments is in the PsaA branch. This
quinone is bound via a single hydrogen bond to the backbone
nitrogen of LeuA722, and the methyl substituent on the
phylloquinone headgroup is meta to the H-bonded carbonyl
(69). This asymmetric H-bonding arrangement distorts the
spin density on the quinone such that it alternates around
the quinone ring and is highest adjacent to the methyl group
(59-61, 70). Because of this, the methyl hyperfine coupling
is unusually large (40, 52, 71) and is partially resolved in
the spin polarization pattern (25, 59-61). In the spectra of
the mutants shown in Figure 4, the methyl hyperfine structure
is not as well resolved, suggesting that the detergent may
affect the strength of the hydrogen bond. LeuA722 is in a
loop region of the protein on the stromal surface of the
membrane but is shielded from the stroma by the peripheral
subunits PsaE and PsaF. Thus, it is possible that, in the
absence of one or both of these subunits, the detergent used
to isolate PS I causes some conformational disorder in the
loop region, leading to a distribution of hydrogen bond
strengths and a distribution of methyl hyperfine couplings.
Simulations (not shown) indicate that the loss of the
shoulders in Figure 4 is consistent with a reduction of the
methyl group hyperfine couplings. However, the strength of
Forward Electron Transfer in Photosystem I
Biochemistry, Vol. 43, No. 5, 2004 1269
FIGURE 5: Left: Calculated spin-polarized EPR spectra of P700+A1(top) and P700+FX- (bottom) for two different lifetimes for P700+A0-.
Solid curves: τ ) 35 ps. Dashed curves: τ ) 1 ns. The spectra
have been calculated as described in detail in refs 62 and 65 and
take into account the spin dynamics in three sequential radial pairs.
The magnetic and geometric parameters are given in ref 65 and
have been taken from EPR experimental data and the X-ray
structure. Right: Experimental spin-polarized EPR spectra of
P700+A1- (top) and P700+FeS- (bottom) of the wild type (solid
curves) and the ∆psaE/∆psaF mutant isolated with Triton X-100
(dashed curves). The spectra of P700+A1- (top) are the signal
intensity at 135 K in a time window centered at 0.6 µs after the
laser flash, while the P700+FeS- spectra (bottom) are taken at room
temperature in a window centered at 1.2 µs (dashed line) after the
laser flash.
the coupling is difficult to assess accurately from the transient
EPR spectra, and further studies (e.g., pulsed ENDOR
measurements (49)) are required to confirm this hypothesis.
The interaction between the subunits and possible structural
changes will be discussed in more detail below.
As mentioned above, the appearance of an absorptive
feature on the high-field side of the P700+FeS- spectrum at
room temperature (Figure 2, dashed curves) is indicative of
singlet-triplet mixing in the state P700+A0- (65). If such
mixing does indeed occur in the mutants, then it should also
lead to changes in the spectra of the subsequent radical pairs
P700+A1- and P700+FX-. In general, any spin polarization
pattern generated by spin dynamics can be broken down into
“net” and “multiplet” contributions. The net contribution
imparts a purely emissive or purely absorptive EPR signal
to each spin, while the multiplet contribution gives each spin
a signal which has equal amounts of absorption and emission.
An extensive discussion of light-induced electron spin
polarization is given in ref 72, and its properties in
photosynthetic reaction centers are reviewed in refs 20 and
25. As discussed in ref 62, under the conditions found in
photosynthetic reaction centers, there are two additional
contributions to the spin polarization induced by singlettriplet mixing in a precursor. The first of these imparts a net
polarization to each of the radicals in subsequent radical pairs
(29). The strength of the polarization is given approximately
by (62)
p)
qb
Ω + k2
2
(1)
where Ω2 ) q2 + b2, q is the difference in the precession
frequencies of the two spins (i.e., the frequency of the
singlet-triplet mixing), b ) 2J + d is the flip-flop term of
the spin-spin coupling, J is the exchange coupling, d is the
dipolar coupling, and k is the mean decay rate for the
precursor radical pair. In reaction centers of heliobacteria,
green sulfur bacteria (64, 65), and modified purple bacteria
(35, 36), this term produces net absorptive polarization of
P+ because both q and b are positive in the initial radical
pair (34-36). Although the sign of b is not known with
certainty for P700+A0- in PS I, it is reasonable to assume
that it is positive as in the other reaction centers. Thus, net
absorptive polarization of P700+ is expected if the lifetime
of P700+A0- is sufficiently long. To analyze the spectrum of
P700+FX-, the generation of the net polarization on both
precursors P700+A0- and P700+A1- must be considered. The
sign of b is opposite in P700+A0- and P700+A1- as a result of
the difference in the relative magnitudes of J and d in the
two radical pairs. Because of this, the contribution to the
net polarization from spin dynamics in P700+A1- is emissive.
Hence, the overall net polarization of P700+ may be of either
sign in P700+FX- depending on the relative strengths of the
absorptive and emissive contributions from the spin dynamics
in P700+A0- and P700+A1-, respectively. In native PS I, the
lifetime of P700+A0- is very short (∼35 ps) so that the
emissive contribution from singlet-triplet mixing in P700+A1dominates the net polarization of P700+ in P700+FX-.
In addition to the net polarization, singlet-triplet mixing
also causes multiplet polarization of the donor because of
the inhomogeneous broadening from unresolved hyperfine
coupling. This term only produces polarization of the donor
because the hyperfine configurations in the two sequential
acceptors are uncorrelated, which leads to cancellation of
the multiplet polarization in the acceptor(s) (26). The
polarization from this term is given by (62)
p)
q∆
Ω + k2
2
(2)
where ∆ is the half-width of the Gaussian line shape
describing inhomgeneous broadening. In PS I, q is positive
and this term produces an E/A (E ) emission, A )
absorption) polarization pattern for P700+. To investigate the
possibility that the changes in the experimental spectra are
related to these effects, the spectra of P700+A1- and P700+FXwere calculated as a function of the lifetime of P700+A0-.
An example of these calculations is shown on the left side
of Figure 5. The solid curves correspond to a P700+A0lifetime of 35 ps, as has been estimated for wild-type PS I
(73), while the dashed curves correspond to a lifetime of 1
ns. The magnetic and geometric parameters used for the
calculations are summarized in Table 1 and have been taken
from the literature. In cases in which several literature values
exist, we have taken an average or chosen one of them.
Although this remaining uncertainty in the parameters does
have some effect on the calculated spectra, the parameters
are sufficiently well established to allow a qualitative analysis
of the effect of an increase in the lifetime of P700+A0-. As
can be seen, the increase in the lifetime leads to the expected
absorbance on the high-field end of the P700+FX- spectrum
(Figure 5, bottom left) and changes the shape of the P700+A1spectrum (Figure 5, top left). On the right side of Figure 5,
the low-temperature spectra of P700+A1- and the room
temperature spectra of P700+FX- in the wild type (solid
1270 Biochemistry, Vol. 43, No. 5, 2004
van der Est et al.
Table 1: Summary of Magnetic and Geometric Parameters Held Fixed in the Simulations
line
g tensors
widths
(mT)
gxx
gyy
gzz
P700+
A0A1FX-
2.0031
2.0031
2.0063
1.78
2.0026
2.0031
2.0051
1.86
2.0022
2.0031
2.0022
2.06
A1- methyl hyperfine coupling (mT)
0.40
0.40
0.18
2.0
refs
61, 76-78
16, 79, 80
37, 38, 61
81
Euler angles (deg) between g(A1) and A(CH3)
Axx
Ayy
Azz
R
β
γ
refs
0.32
0.32
0.46
30
0
0
49, 53, 61
Euler angles (deg) relating g tensors
P700+ A0P700+ A1P700+ FX-
dipolar axis in g(P700)
spin-spin coupling (mT)
R
β
γ
θ
φ
J
D
refs
2.0
180.0
126.0
131.1
81.0
70.6
17.9
34.5
59.7
153.2
78.7
65.4
0.5
0.0
0.0
-0.34
-0.17
-0.10
65, 69
44, 61, 65, 82
65, 69
FIGURE 6: Experimental data (dashed lines) and simulations (solid
lines) of the transient EPR data sets for the PS I complexes isolated
with Triton X-100. Representative boxcar spectra are shown in three
different time windows centered at the times indicated. The
procedure used to calculate the simulated spectra is discussed in
the text.
FIGURE 7: Experimental data (dashed lines) and simulations (solid
lines) of the transient EPR data sets for the PS I complexes isolated
with β-DM. Representative boxcar spectra are shown in three
different time windows.
curves) and ∆psaE/∆psaF mutant (dashed curves) are
compared for Triton X-100 complexes. Clearly the changes
in the experimental spectra resemble those in the simulations.
Thus, it is likely that forward electron transfer from A0- to
A1 is also affected in the mutants, particularly when Triton
X-100 is used in the isolation of PS I.
Analysis of Room Temperature Transient EPR Data.
Figures 6 and 7 show the results of fits to the room
temperature data sets based on a kinetic model in which two
populations of reaction centers are assumed. The EPR signal
S(t,B0) ) (1 - X)S1(t,B0) + XS2(t,B0)
as a function of field position and time is then given by
S1(t,B0) ) R(B0)e-(k1+w)t + β(B0)e-wt(1 - e-k1t)
S2(t,B0) ) R′(B0)e-(k1′+w)t + β′(B0)e-wt(1 - e-k1′t)
(3)
where k1 and k1′ are the rates of electron transfer from A1to FX and X is the fraction of reaction centers in which the
forward electron transfer is governed by k1′. The rate of spin
Forward Electron Transfer in Photosystem I
Biochemistry, Vol. 43, No. 5, 2004 1271
Table 2: Kinetic Parameters and Amplitudes Obtained from Global
Fits of the Transient EPR Data Setsa
detergent
sample
k-1
(ns)
w-1
(µs)
X
Triton X-100
WT
∆psaE
∆psaF
∆psaE/∆psaF
WT
∆psaE
∆psaF
∆psaE/∆psaF
340
300
250
210
290
320
320
340
1.04
1.23
1.54
1.38
1.25
1.25
1.27
1.33
0.17
0.23
0.35
0.55
0.17
0.20
0.19
0.36
∆-DM
a
The parameters k, w, and X are defined in eq 3.
relaxation is given by w and is assumed to be the same for
all radical pairs. The spectra of P700+A1- and P700+FX- for
the two fractions are represented by R(B0), R′(B0), β(B0), and
β′(B0). Here, we calculate the spectra of these species and
fit S(t,B0) to the time/field data sets with k1, w, and X as free
parameters. The rise time of the spectrometer and the lifetime
broadening are taken into account by convoluting S(t,B0) with
the spectrometer response function and a Lorentzian line
shape, respectively. With these assumptions, we have
separated the spectral dependence and time dependence of
the data and have ignored coherent oscillations (55, 74, 75)
of the spin system. We expect this approximation to be valid
for times that are long compared to the lifetime of singlettriplet mixing in the sequential radical pairs, i.e., times longer
than ∼50 ns, but not for the short time behavior of the
system. Thus, we can use this approach to reproduce the
spectra at various times >50 ns and estimate the fraction of
fast electron transfer. It is important to point out that the
values of the parameters obtained from the fits can be
expected to show trends in the data but the ratio of the two
kinetic components will have a large uncertainty associated
with it because of the assumptions involved.
For the fits shown in Figures 6 and 7, we have used the
geometric and magnetic parameters given in Table 1 and
have assumed that the two fractions have different electrontransfer kinetics. In one fraction (corresponding to S1 in eq
3), the lifetime of A0- is assumed to be too short for
significant singlet-triplet mixing to occur (i.e., <∼500 ps),
and the lifetime of A1- (k1) is an adjustable parameter, which
we expect to yield values of ∼250 ns. In the second fraction,
which corresponds to S2 in eq 3 and X in Table 2, we
arbitrarily assume that electron transfer from A0- to A1
occurs with a lifetime of 1 ns. This is based on the fact that
changes similar to those observed experimentally are expected for A0- lifetimes in this range. The second fraction
is further assumed to correspond to the fast component of
electron transfer from A1- to FX with a lifetime of 10 ns. As
can be seen in the two figures, the experimental spectra are
reproduced very well by this procedure, and it yields lifetimes
that are in good agreement with previously determined values
(11). The values of the fitted parameters are given in Table
2. The fraction of fast electron transfer in the wild type is
∼20%, in reasonable agreement with the ∼25-30% determined optically (4, 14), and the changes in the spectra are
reproduced well by increasing the fraction of fast electron
transfer. However, these values should be treated with caution
because three of the lifetimes used are below the time
resolution of the spectrometer, and we have arbitrarily
assumed values for them. Fits with different assumed values
for the lifetimes give different ratios of the two phases, but
the overall trends remain the same. For example, smaller
values of X are obtained if a shorter lifetime for A0- or a
longer lifetime for A1- is assumed for this fraction. It is also
unlikely that the reoxidation of A1- is strictly biphasic, but
rather the two lifetimes are probably the major components
in a distribution of lifetimes. Indeed, there is evidence for
this from recent low-temperature optical studies (14). Thus,
the fits give only a qualitative estimate of how the fast
component changes. Despite these limitations, it is clear from
the analysis that the fraction of fast electron transfer to FX
is increased by the combined effects of the mutations and
detergent isolation and the effect is largest in the ∆PsaE/
∆PsaF mutant.
DISCUSSION
The results presented above show that isolation of PS I
with Triton X-100 has a significant influence on electron
transfer from A1 to FX when the PsaF subunit is absent, and
this effect is enhanced when PsaE is also absent. This mirrors
almost exactly the photoaccumulation experiments in PS I
mutants, in which a population of A0- was photoaccumulated
at the expense of A1- when the PsaF subunit was missing
and Triton X-100 was employed, and this effect was similarly
enhanced when PsaE was also absent (16). The larger fraction
of fast electron transfer in the Triton X-100 preparations is
also reminiscent of similar effects observed in spinach
complexes (8). Indeed, the transient EPR data from spinach
PS Iβ complexes (25) closely resemble those of the Triton
X-100 complexes of the double mutant presented in Figures
1 and 2 (bottom left). As discussed above, the proposal that
the biphasic reoxidation of A1- represents electron transfer
in the two branches of cofactors implies that changes in the
ratio of the two phases represent changes in the efficiency
of electron transfer in the two branches. Indeed, this
assumption is important to the interpretation of the spectroscopic data of samples with point mutations which are
expected to alter the quantum yield of electron transfer, e.g.,
in the A0 binding site or in the vicinity of P700. The results
presented here suggest that caution should be exercised in
interpreting changes in the ratio of the two kinetic phases.
The spectra in Figure 4, which are known to be associated
with the PsaA branch quinone, are also altered, and there is
evidence that forward electron transfer from A0- may also
be slowed in the PsaA branch. Both of these effects are
consistent with an alteration of the environment and properties of QK-A. That such changes should occur is not
surprising given the locations of PsaE and PsaF on the
stromal side of the PS I complex near the PsaA branch
electron transport cofactors (Figure 1). The contacts between
PsaF and other subunits of the PS I complex are listed in
Table 3. As shown in the left column of Table 3, PsaF is
hydrogen bonded to PsaA, PsaB, and PsaE. In the last two
columns, the locations of the contacts within the complex
are given. From this it is clear that the majority of the
contacts are on the stromal side of the membrane, primarily
in the A-jk(1) loop/helix region. A smaller series of contacts
is also found in the B-gh loop region on the lumenal side of
the membrane. However, these are well removed from the
electron-transfer cofactors, which are bound via residues
predominantly in helix j. Moreover, the changes in the
electron-transfer kinetics are enhanced by removal of PsaE,
1272 Biochemistry, Vol. 43, No. 5, 2004
van der Est et al.
Table 3: Contacts between PsaF and Other PS I Subunits
PsaF
distance
subunit residue atom residue atom
(Å)
PsaA
PsaA
PsaA
PsaA
PsaA
PsaA
PsaA
PsaA
PsaB
PsaB
PsaB
PsaB
PsaB
PsaB
PsaB
PsaB
PsaB
PsaE
PsaE
PsaE
Lys710
Lys710
Leu711
Lys712
Lys712
Ala714
Ala716
Ile717
Arg548
Glu419
Lys418
Gln416
Lys551
Glu459
Glu459
Leu457
Glu453
Pro12
H2O152
Tyr17
O
Nζ
O
O
O
O
N
N
Nη1
O1
Nζ
O
Nζ
N
O2
O
O1
O
O
O
Ala131
Asp133
Arg82
Arg89
Arg89
Arg89
Glu98
Glu98
Arg141
Arg141
Ser129
Arg141
Thr137
Leu51
His50
Leu51
Arg34
H2O152
Val138
Thr37
N
Oδ2
N
Nη1
Nη2
Nη2
O2
O1
Oξt
Nη2
Oγ
Nη2
Oγ1
O
Nδ1
N
N
O
O
Oγ1
3.02
2.73
3.03
3.08
2.88
3.16
2.83
2.99
2.76
2.58
2.63
2.99
3.16
3.28
2.40
2.79
2.64
3.11
3.12
2.83
side
location
stroma
stroma
stroma
stroma
stroma
stroma
stroma
stroma
stroma
stroma
stroma
stroma
stroma
lumen
lumen
lumen
lumen
stroma
stroma
stroma
A-jk(1) helix
A-jk(1) helix
A-jk(1) helix
A-jk(1) helix
A-jk(1) helix
A-jk(1) loop
A-jk(1) loop
A-jk(1) loop
B-h helix
B-g helix
B-g-helix
B-fg loop
B-hi loop
B-gh loop
B-gh loop
B-gh loop
B-gh loop
which is located on the stromal side of the complex. Thus,
it is unlikely that changes near the contacts on the lumenal
side of PS I are responsible for the observed effects. The
contacts in the A-jk(1) loop/helix region are depicted in more
detail in the top part of Figure 8, which shows a view roughly
along the membrane normal. As enumerated in Table 3, PsaF
is bound to PsaA through a series of eight hydrogen bonds
to residues between K710PsaA and I717PsaA. Thus, it appears
that one of the roles of PsaF is to stabilize the structure of
the A-jk(1) loop. This has obvious importance for electron
transfer in PS I because the quinone in the PsaA branch (QKA) is hydrogen bonded to the backbone nitrogen of L722PsaA,
which is located near the region in contact with PsaF. In the
absence of PsaF, it is likely that conformational changes
occur in the A-jk(1) loop, which in turn alter the properties
of QK-A. The contacts to PsaE are also located in this region
so that any conformational changes resulting from the loss
of PsaF would be enhanced in the absence of PsaE. In ref
16, it was postulated that the action of Triton X-100 was to
open a water channel to the quinone, allowing it to be more
easily protonated and doubly reduced. Such a pathway is
apparent in the 2.5 Å crystal structure if one postulates that
Triton X-100 is able to remove chlorophyll cofactors in this
region when PsaF is missing. In the bottom part of Figure
8, the A-jk(1) loop region is shown roughly along the
membrane plane. As can be seen, chlorophylls 1138 and 1139
are bound near the stromal surface in close proximity to both
PsaF and the A-jk(1) loop. A series of tentatively identified
water molecules are also shown, and it appears that the two
chlorophylls shield QK-A from at least two of them. Since it
is possible that Triton X-100 can extract one or both of the
chlorophyll molecules in the absence of PsaF, this could
expose QK-A to stromal water. Such changes would also be
expected to influence the H-bonding between QK-A and
L722PsaA, which would be reflected in changes in the spin
density distribution and the hyperfine couplings. Similarly,
it is well-known that the redox potential of A1 is strongly
influenced by the surrounding protein so that changes in the
binding of the quinone alter the electron-transfer kinetics.
Indeed, this is the basis of the observations that point
mutations in the A1 binding site affect the electron-transfer
FIGURE 8: Structural features near PsaF on the stromal side of PS
I from the 2.5 Å resolution X-ray structure (69) (PDB entry 1JB0).
Top: View of the A-jk(1) loop region from the stromal side of the
membrane roughly along the membrane normal. H-bonding contacts
between PsaA and PsaF are shown. Bottom: The same region is
shown roughly parallel to the membrane plane. Two chlorophyll
molecules (chl 1138 and chl 1139), which may be susceptible to
solubilization in the absence of PsaF, are shown. The presence of
water molecules on the stromal side of the chlorophylls suggests
that QK-A would be exposed to water if these chlorophylls were
extracted by Triton-X-100. The figure was constructed using the
program MOLMOL (83).
kinetics. Although none of our data show conclusively in
which branch the fast component of electron transfer induced
by the absence of PsaF and Triton X-100-mediated changes
occur, the most structurally reasonable explanation is that it
is the PsaA branch. The alternative explanation that the
change in the ratio of the fast and slow kinetic components
results from a greater fraction of electron transfer in the PsaB
branch would require that the initial ratio of charge separation
is altered in the two branches. None of the contacts shown
in Table 3 are close to P700 or A0, and thus, it is difficult to
see how changes associated with the absence of PsaF could
influence the directionality. Moreover, this explanation would
require that double reduction of the quinone in the PsaA
branch takes place while photoaccumulation of A1- does not
occur in the PsaB branch (16).
Recently an interesting alternative model for biphasic
kinetic behavior was suggested (14) in which the electron
transfer was proposed to proceed along the PsaB branch to
FX with a lifetime of 10 ns and then to either FA/FB or QK-A
with a faster, unspecified lifetime. The slow phase of A1reoxidation would then correspond to electron transfer from
Qk-A via FX to FA/FB. Under this model a change in the ratio
of the two phases would represent a change in the fraction
of electrons being transferred from FX- to FA/FB or QK-A.
Such a change would also be expected if the structural
changes outlined above occurred. However, this model is
Forward Electron Transfer in Photosystem I
difficult to reconcile with the spin-polarized EPR data
because the fast electron spin relaxation of FX- and spin
evolution in the radical pair P700+QK-B- should lead to
P700+QK-A- spectra which are different from those observed.
Moreover, removal of FX should also lead to changes in the
spin polarization patterns, yet no such differences are seen
(68).
Thus, the data presented here suggest that structural
changes promoted by the removal of PsaF induce fast
reoxidation of the quinone in the PsaA branch and that this
effect is probably responsible for the changes in the ratio of
the two kinetic phases in spinach preparations. However, it
is important to point out that although this explanation of
the detergent-induced effects is more in line with the
unidirectional model, it does not rule out a bidirectional
model in intact PS I.
REFERENCES
1. Boudreaux, B., MacMillan, F., Teutloff, C., Agalarov, R., Gu, F.
F., Grimaldi, S., Bittl, R., Brettel, K., and Redding, K. (2001)
Mutations in both sides of the Photosystem I reaction center
identify the phylloquinone observed by electron paramagnetic
resonance spectroscopy, J. Biol. Chem. 276, 37299-37306.
2. Guergova-Kuras, M., Boudreaux, B., Joliot, A., Joliot, P., and
Redding, K. (2001) Evidence for two active branches for electron
transfer in Photosystem I, Proc. Natl. Acad. Sci. U.S.A. 98, 44374442.
3. Muhiuddin, I. P., Heathcote, P., Carter, S., Purton, S., Rigby, S.
E. J., and Evans, M. C. W. (2001) Evidence from time resolved
studies of the P700+A1- radical pair for photosynthetic electron
transfer on both the PsaA and PsaB branches of the Photosystem
I reaction centre, FEBS Lett. 503, 56-60.
4. Xu, W., Chitnis, P., Valieva, A., van der Est, A., Brettel, K.,
Guergova-Kuras, M., Pushkar, J., Zech, S., Stehlik, D., Shen, G.,
Zybailov, B., and Golbeck, J. (2003) Electron transfer in cyanobacterial Photosystem I. II. Determination of forward electron
transfer rates of site-directed mutants in a putative electron transfer
pathway from A0 through A1 to FX, J. Biol. Chem. 278, 2787627887.
5. Xu, W., Chitnis, P., Valieva, A., van der Est, A., Pushkar, J.,
Krzystyniak, M., Teutloff, C., Zech, S. G., Bittl, R., Stehlik, D.,
Zybailov, B., Shen, G., and Golbeck, J. (2003) Electron transfer
in cyanobacterial Photosystem I. I. Physiological and spectroscopic
characterization of site-directed mutants in a putative electron
transfer pathway from A0 through A1 to FX, J. Biol. Chem. 278,
27864-27875.
6. Brettel, K. (1997) Electron transfer and arrangement of the redox
cofactors in Photosystem I, Biochim. Biophys. Acta 1318, 322373.
7. Brettel, K., and Leibl, W. (2001) Electron transfer in Photosystem
I, Biochim. Biophys. Acta 1507, 100-114.
8. Sétif, P., and Brettel, K. (1993) Forward electron transfer from
phylloquinone A1 to iron-sulfur centers in spinach Photosystem
I, Biochemistry 32, 7846-7854.
9. Brettel, K. (1988) Electron transfer from A1- to an iron-sulfur
center with t1/2 ) 200ns at room temperature in Photosystem I,
FEBS Lett. 239, 93-98.
10. Bock, C. H., van der Est, A. J., Brettel, K., and Stehlik, D. (1989)
Nanosecond electron transfer kinetics in Photosystem I as obtained
from transient EPR at room temperature, FEBS Lett. 247, 9196.
11. van der Est, A., Bock, C., Golbeck, J., Brettel, K., Sétif, P., and
Stehlik, D. (1994) Electron transfer from acceptor A1 to the ironsulfur centers in Photosystem I as studied by transient EPR
spectroscopy, Biochemistry 33, 11789-11797.
12. Brettel, K. (1999) Electron transfer from acceptor A1 to the ironsulfur clusters in Photosystem I measured with a time resolution
of 2 ns, in Photosynthesis, Mechanisms and Effects (Garab, G.,
Ed.) pp 611-614, Kluwer Academic, Dordrecht, The Netherlands.
13. Schlodder, E., Falkenberg, K., Gergeleit, M., and Brettel, K. (1998)
Temperature dependence of forward and reverse electron transfer
from A1-, the reduced secondary electron acceptor in Photosystem
I, Biochemistry 37, 9466-9476.
Biochemistry, Vol. 43, No. 5, 2004 1273
14. Agalarov, R., and Brettel, K. (2003) Temperature dependence of
biphasic forward electron transfer from the phylloquinone(s) A1
in Photosystem I: only the slower phase is activated, Biochim.
Biophys. Acta, Bioenerg. 1604, 7-12.
15. Jolliot, P., and Jolliot, A. (1999) In vivo analysis of the electron
transfer within Photosystem I: Are the two phylloquinones
Involved? Biochemistry 38, 11130-11136.
16. Yang, F., Shen, G. Z., Schluchter, W. M., Zybailov, B. L., Ganago,
A. O., Vassiliev, I. R., Bryant, D. A., and Golbeck, J. H. (1998)
Deletion of the PsaF polypeptide modifies the environment of the
redox-active phylloquinone A1. Evidence for unidirectionality of
electron transfer in Photosystem I, J. Phys. Chem. B 102, 82888299.
17. Schubert, W. D., Klukas, O., Krauss, N., Saenger, W., Fromme,
P., and Witt, H. T. (1997) Photosystem I of Synechococcus
elongatus at 4 Å resolution: comprehensive structure analysis, J.
Mol. Biol. 272, 741-769.
18. Lagoutte, B., Sétif, P., and Duranton, J. (1984) Tentative identification of the apoproteins of iron-sulfur centers of Photosystem
I, FEBS Lett. 174, 24-29.
19. Hoff, A. (1993) In The Photosynthetic Reaction Center (Deisenhofer, J., and Norris, J. R., Eds.) pp 331-386, Academic Press,
San Diego/London.
20. Snyder, S. W., and Thurnauer, M. (1993) In The Photosynthetic
Reaction Center (Deisenhofer, J., and Norris, J. R., Eds.) pp 285330, Academic Press, San Diego/London.
21. Angerhofer, A., and Bittl, R. (1996) Radicals and radical pairs in
photosynthesis, Photochem. Photobiol. 63, 11-38.
22. Möbius, K. (2001) High-field and high-frequency electron paramagnetic resonance, Appl. Magn. Reson. 21, 255-255.
23. Möbius, K. (2000) Primary processes in photosynthesis: what do
we learn from high-field EPR spectroscopy? Chem. Soc. ReV. 29,
129-139.
24. Stehlik, D., and Möbius, K. (1997) New EPR methods for
investigating photoprocesses with paramagnetic intermediates,
Annu. ReV. Phys. Chem. 48, 745-784.
25. van der Est, A. (2001) Light-induced spin polarization in type I
photosynthetic reaction centres, Biochim. Biophys. Acta 1507,
212-225.
26. Pedersen, J. B. (1979) Determination of the primary reactions of
photosynthesis from transient ESR signals, FEBS Lett. 97, 305310.
27. Norris, J. R., Morris, A. L., Thurnauer, M. C., and Tang, J. (1990)
A general model of electron-spin polarization arising from the
interactions within radical pairs, J. Chem. Phys. 92, 4239-4249.
28. Morris, A. L., Snyder, S. W., Zhang, Y. N., Tang, J., Thurnauer,
M. C., Dutton, P. L., Robertson, D. E., and Gunner, M. R. (1995)
Electron-spin polarization model applied to sequential electrontransfer in iron-containing photosynthetic bacterial reaction centers
with different quinones as QA, J. Phys. Chem. 99, 3854-3866.
29. Hore, P. J. (1996) Transfer of spin correlation between radical
pairs in the initial steps of photosynthetic energy conversion, Mol.
Phys. 89, 1195-1202.
30. Kandrashkin, Y. E., Salikhov, K. M., and Stehlik, D. (1997) Spin
dynamics and EPR spectra of consecutive spin-correlated radical
pairs. Model calculations, Appl. Magn. Reson. 12, 141-166.
31. Snyder, S. W., Morris, A. L., Bondeson, S. R., Norris, J. R., and
Thurnauer, M. C. (1993) Electron-spin polarization in sequential
electron transfersan example from iron-containing photosynthetic
bacterial reaction center proteins, J. Am. Chem. Soc. 115, 37743775.
32. Utschig, L. M., Greenfield, S. R., Tang, J., Laible, P. D., and
Thurnauer, M. C. (1997) Influence of iron-removal procedures
on sequential electron transfer in photosynthetic bacterial reaction
centers studied by transient EPR spectroscopy, Biochemistry 36,
8548-8558.
33. Tang, J., Bondeson, S., and Thurnauer, M. C. (1996) Effects of
sequential electron transfer on electron spin polarized transient
EPR spectra at high fields, Chem. Phys. Lett. 253, 293-298.
34. Tang, J., Utschig, L. M., Poluektov, O., and Thurnauer, M. C.
(1999) Transient W-band EPR study of sequential electron transfer
in photosynthetic bacterial reaction centers, J. Phys. Chem. B 103,
5145-5150.
35. Hulsebosch, R. J., Borovykh, I. V., Paschenko, S. V., Gast, P.,
and Hoff, A. J. (1999) Radical pair dynamics and interactions in
quinone-reconstituted photosynthetic reaction centers of Rb.
sphaeroides R26: A multifrequency magnetic resonance study,
J. Phys. Chem. B 103, 6815-6823.
1274 Biochemistry, Vol. 43, No. 5, 2004
36. Hulsebosch, R. J., Borovykh, I. V., Paschenko, S. V., Gast, P.,
and Hoff, A. (2001) Erratum: radical pair dynamics and interactions in quinone-reconstituted photosynthetic reaction centers of
Rb. Sphaeroides R26: A multifrequency magnetic resonance
study, J. Phys. Chem. B 105, 10146.
37. van der Est, A., Prisner, T., Bittl, R., Fromme, P., Lubitz, W.,
Möbius, K., and Stehlik, D. (1997) Time-resolved X-, K-, and
W-band EPR of the radical pair state P700+A1- of Photosystem I
in comparison with P865+QA- in bacterial reaction centers, J. Phys.
Chem. B 101, 1437-1443.
38. MacMillan, F., Hanley, J., van der Weerd, L., Knupling, M., Un,
S., and Rutherford, A. W. (1997) Orientation of the phylloquinone
electron acceptor anion radical in Photosystem I, Biochemistry
36, 9297-9303.
39. Berthold, T., Bechtold, M., Heinen, U., Link, G., Poluektov, O.,
Utschig, L., Tang, J., Thurnauer, M. C., and Kothe, G. (1999)
Magnetic-field-induced orientation of photosynthetic reaction
centers as revealed by time-resolved W-band EPR of spincorrelated radical pairs, J. Phys. Chem. B 103, 10733-10736.
40. Teutloff, C., Hofbauer, W., Zech, S. G., Stein, M., Bittl, R., and
Lubitz, W. (2001) High-frequency EPR studies on cofactor radicals
in Photosystem I, Appl. Magn. Reson. 21, 363-379.
41. Fuhs, M., Schnegg, A., Prisner, T., Kohne, I., Hanley, J.,
Rutherford, A. W., and Möbius, K. (2002) Orientation selection
in photosynthetic PS I multilayers: structural investigation of the
charge separated state P700+A1- by high-field/high-frequency timeresolved EPR at 3.4 T/95 GHz, Biochim. Biophys. Acta 1556, 8188.
42. Zech, S. G., Lubitz, W., and Bittl, R. (1996) Pulsed EPR
experiments on radical pairs in photosynthesis: Comparison of
the donor-acceptor distances in Photosystem I and bacterial
reaction centers, Ber. Bunsen-Ges. Phys. Chem. 100, 2041-2044.
43. Bittl, R., Zech, S. G., Fromme, P., Witt, H. T., and Lubitz, W.
(1997) Pulsed EPR structure analysis of photosystem I single
crystals: localization of the phylloquinone acceptor, Biochemistry
36, 12001-12004.
44. Zech, S. G., van der Est, A. J., and Bittl, R. (1997) Measurement
of cofactor distances between P700+ and A1- in native and quinonesubstituted Photosystem I using pulsed electron paramagnetic
resonance spectroscopy, Biochemistry 36, 9774-9779.
45. Bittl, R., and Zech, S. G. (1997) Pulsed EPR study of spin-coupled
radical pairs in photosynthetic reaction centers: Measurement of
the distance between P700+ and A1- in Photosystem I and between
P865+ and QA- in bacterial reaction centers, J. Phys. Chem. B 101,
1429-1436.
46. Dzuba, S. A., Hara, H., Kawamori, A., Iwaki, M., Itoh, S., and
Tsvetkov, Y. D. (1997) Electron spin echo of spin-polarised radical
pairs in intact and quinone-reconstituted plant Photosystem I
reaction centres, Chem. Phys. Lett. 264, 238-244.
47. Iwaki, M., Itoh, S., Hara, H., and Kawamori, A. (1998) Spinpolarized radical pair in Photosystem I reaction center that contains
different quinones and fluorenones as the secondary electron
acceptor, J. Phys. Chem. B 102, 10440-10445.
48. Bittl, R., and Zech, S. G. (2001) Pulsed EPR spectroscopy on shortlived intermediates in photosystem I, Biochim. Biophys. Acta 1507,
194-211.
49. Fursman, C. E., Teutloff, C., and Bittl, R. (2002) Pulsed ENDOR
studies of short-lived spin-correlated radical pairs in photosynthetic
reaction centers, J. Phys. Chem. B 106, 9679-9686.
50. Käss, H., Fromme, P., Witt, H. T., and Lubitz, W. (2001)
Orientation and electronic structure of the primary donor radical
cation P700+ in Photosystem I: A single crystals EPR and ENDOR
study, J. Phys. Chem. B 105, 1225-1239.
51. Mino, H., Kawamori, A., Itoh, K., Miyamoto, R., and Itoh, S.
(2003) Pulsed ENDOR study of spin-correlated radical pair in
Photosystem I, Plant Cell Physiol. 44, S156-S156.
52. Muhiuddin, I. P., Rigby, S. E. J., Evans, M. C. W., Amesz, J.,
and Heathcote, P. (1999) ENDOR and special TRIPLE resonance
spectroscopy of photoaccumulated semiquinone electron acceptors
in the reaction centers of green sulfur bacteria and heliobacteria,
Biochemistry 38, 7159-7167.
53. Rigby, S. E. J., Evans, M. C. W., and Heathcote, P. (1996) ENDOR
and special triple resonance spectroscopy of A1- of Photosystem
I, Biochemistry 35, 6651-6656.
54. Rigby, S. E. J., Evans, M. C. W., and Heathcote, P. (2001) Electron
nuclear double resonance (ENDOR) spectroscopy of radicals in
Photosystem I and related Type 1 photosynthetic reaction centres,
Biochim. Biophys. Acta 1507, 247-259.
van der Est et al.
55. Kothe, G., Weber, S., Bittl, R., Ohmes, E., Thurnauer, M. C., and
Norris, J. R. (1991) Transient EPR of light-induced radical pairs
in plant Photosystem-Isobservation of quantum beats, Chem.
Phys. Lett. 186, 474-480.
56. Weber, S., Ohmes, E., Thurnauer, M. C., Norris, J. R., and Kothe,
G. (1995) Light-generated nuclear quantum beatssa signature of
photosynthesis, Proc. Natl. Acad. Sci. U.S.A. 92, 7789-7793.
57. Kamlowski, A., van der Est, A., Fromme, P., and Stehlik, D. (1997)
Low temperature EPR on Photosystem I single crystals: Orientation of the iron-sulfur centers FA and FB, Biochim. Biophys. Acta,
Bioenerg. 1319, 185-198.
58. Kamlowski, A., van der Est, A., Fromme, P., Krauss, N., Schubert,
W. D., Klukas, O., and Stehlik, D. (1997) The structural
organization of the PsaC protein in Photosystem I from single
crystal EPR and X-ray crystallographic studies, Biochim. Biophys.
Acta 1319, 199-213.
59. Kamlowski, A., Zech, S. G., Fromme, P., Bittl, R., Lubitz, W.,
Witt, H. T., and Stehlik, D. (1998) The radical pair state P700+A1in Photosystem I single crystals: Orientation dependence of the
transient spin-polarized EPR spectra, J. Phys. Chem. B 102, 82668277.
60. Kamlowski, A., Altenberg-Greulich, B., van der Est, A., Zech, S.
G., Bittl, R., Fromme, P., Lubitz, W., and Stehlik, D. (1998) The
quinone acceptor A1 in Photosystem I: Binding site, and
comparison to QA in purple bacteria reaction centers, J. Phys.
Chem. B 102, 8278-8287.
61. Zech, S. G., Hofbauer, W., Kamlowski, A., Fromme, P., Stehlik,
D., Lubitz, W., and Bittl, R. (2000) A structural model for the
charge separated state P700+A1- in Photosystem I from the
orientation of the magnetic interaction tensors, J. Phys. Chem. B
104, 9728-9739.
62. Kandrashkin, Y. E., Salikhov, K. M., van der Est, A., and Stehlik,
D. (1998) Electron spin polarization in consecutive spin-correlated
radical pairs: Application to short-lived and long-lived precursors
in type 1 photosynthetic reaction centres, Appl. Magn. Reson. 15,
417-447.
63. Kandrashkin, Y., and van der Est, A. (2002) Electron spin
polarization in photosynthetic reaction centres: Strategies for
extracting structural and functional information, Riken ReV. 44,
124-127.
64. van der Est, A., Hager-Braun, C., Leibl, W., Hauska, G., and
Stehlik, D. (1998) Transient electron paramagnetic resonance
spectroscopy on green-sulfur bacteria and heliobacteria at two
microwave frequencies, Biochim. Biophys. Acta, Bioenerg. 1409,
87-98.
65. Kandrashkin, Y. E., Vollmann, W., Stehlik, D., Salikhov, K., and
van der Est, A. (2002) The magnetic field dependence of the
electron spin polarization in consecutive spin correlated radical
pairs in type I photosynthetic reaction centres, Mol. Phys. 100,
1431-1443.
66. Sakuragi, Y., Zybailov, B., Shen, G. Z., Jones, A. D., Chitnis, P.
R., van der Est, A., Bittl, R., Zech, S., Stehlik, D., Golbeck, J.
H., and Bryant, D. A. (2002) Insertional inactivation of the menG
gene, encoding 2-phytyl- 1,4-naphthoquinone methyltransferase
of Synechocystis sp PCC 6803, results in the incorporation of
2-phytyl-1,4-naphthoquinone into the A1 site and alteration of the
equilibrium constant between A1 and Fx in Photosystem I,
Biochemistry 41, 394-405.
67. Moenne Loccoz, P., Heathcote, P., Maclachlan, D. J., Berry, M.
C., Davis, I. H., and Evans, M. C. W. (1994) Path of electron
transfer in Photosystem 1sDirect evidence of forward electron
transfer from A1 to Fe-Sx, Biochemistry 33, 10037-10042.
68. Shen, G. Z., Antonkine, M. L., van der Est, A., Vassiliev, I. R.,
Brettel, K., Bittl, R., Zech, S. G., Zhao, J. D., Stehlik, D., Bryant,
D. A., and Golbeck, J. H. (2002) Assembly of Photosystem I. II.
Rubredoxin is required for the in vivo assembly of Fx in
Synechococcus sp PCC 7002 as shown by optical and EPR
spectroscopy, J. Biol. Chem. 277, 20355-20366.
69. Jordan, P., Fromme, P., Witt, H. T., Klukas, O., Saenger, W., and
Krauss, N. (2001) Three-dimensional structure of cyanobacterial
Photosystem I at 2.5 angstrom resolution, Nature 411, 909-917.
70. O’Malley, P. J. (1999) Density functional calculated spin densities
and hyperfine couplings for hydrogen bonded 1,4-naphthosemiquinone and phyllosemiquinone anion radicals: a model for the
A1 free radical formed in Photosystem I, Biochim. Biophys. Acta
1411, 101-113.
71. Fursman, C. E., Bittl, R., Zech, S. G., and Hore, P. J. (2001) 95
GHz ESEEM of radical pairs: a source of radical separations and
relative orientations, Chem. Phys. Lett. 342, 162-168.
Forward Electron Transfer in Photosystem I
72. Salikhov, K. M., Molin, Y. N., Sagdeev, R. Z., and Buchachenko,
A. L. (1984) Spin polarization and magnetic effects in radical
reactions, Elsevier, Amsterdam.
73. Brettel, K., and Vos, M. H. (1999) Spectroscopic resolution of
the picosecond reduction kinetics of the secondary electron
acceptor A1 in Photosystem I, FEBS Lett. 447, 315-317.
74. Bittl, R., and Kothe, G. (1991) Transient EPR of radical pairs in
photosynthetic reaction centerssPrediction of quantum beats,
Chem. Phys. Lett. 177, 547-553.
75. Weber, S., Berthold, T., Ohmes, E., Thurnauer, M. C., Norris, J.
R., and Kothe, G. (1996) Nuclear coherences in photosynthetic
reaction centers following light excitation, Appl. Magn. Reson.
11, 461-469.
76. Poluektov, O. G., Utschig, L. M., Schlesselman, S. L., Lakshmi,
K. V., Brudvig, G. W., Kothe, G., and Thurnauer, M. C. (2002)
Electronic structure of the P700 special pair from high-frequency
electron paramagnetic resonance spectroscopy, J. Phys. Chem. B
106, 8911-8916.
77. Prisner, T. F., McDermott, A. E., Un, S., Norris, J. R., Thurnauer,
M. C., and Griffin, R. G. (1993) Measurement of the G-tensor of
the P700+ signal from deuterated cyanobacterial Photosystem I
particles, Proc. Natl. Acad. Sci. U.S.A. 90, 9485-9488.
78. Bratt, P. J., Rohrer, M., Krzystek, J., Evans, M. C. W., Brunel, L.
C., and Angerhofer, A. (1997) Submillimeter high-field EPR
Biochemistry, Vol. 43, No. 5, 2004 1275
studies of the primary donor in plant Photosystem I P700, J. Phys.
Chem. B 101, 9686-9689.
79. McCracken, J. L., and Sauer, K. (1983) Orientation dependence
of radical pair interactions in spinach chloroplasts, Biochim.
Biophys. Acta 724, 83-93.
80. Bonnerjea, J., and Evans, M. C. W. (1982) Identification of
multiple components in the intermediary electron carrier complex
of Photosystem I, FEBS Lett. 148, 313-316.
81. Guigliarelli, B., Guillaussier, J., More, C., Sétif, P., Bottin, H.,
and Bertrand, P. (1993) Structural organization of the iron-sulfur
centers in Synechocystis 6803 Photosystem I. EPR study of
oriented thylakoid membranes and analysis of the magnetic
interactions, J. Biol. Chem. 268, 900-8.
82. Link, G., Berthold, T., Bechtold, M., Weidner, J. U., Ohmes, E.,
Tang, J., Poluektov, O., Utschig, L., Schlesselman, S. L.,
Thurnauer, M. C., and Kothe, G. (2001) Structure of the P700+A1radical pair intermediate in Photosystem I by high time resolution
multifrequency electron paramagnetic resonance: Analysis of
quantum beat oscillations, J. Am. Chem. Soc. 123, 4211-4222.
83. Koradi, R., Billeter, M., and Wuthrich, K. (1996) MOLMOL: a
program for display and analysis of macromolecular structures,
J. Mol. Graphics 14, 51-5, 29-32.
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